Global impact of CMP-001 on various immune cell subsets

Purpose: The goal of this study was to investigate the impact of CMP-001 on gene expression patterns within various immune cell subsets. Methods: Unfractionated PBMCs from a healthy donor were isolated via ficol gradient and treated with and without anti-Qbeta coated CMP-001 (10ug/ml) for 24 hours. Untreated and treated fractions were sequenced on an Illumina HiSeq4000 system. FASTQ files were generated from basecall files with the bcl2fastq software (Illumina) provided by the University of Iowa Institute of Human Genetics. Data files were subsequently downloaded to Argon and mapped to the prebuilt GRCh38 genome with CellRanger (version 3.0.1). After initial mapping datasets were merged together and clustering was performed based on merged data sets using Cell Ranger software. Cells with unique gene counts fewer than 300 or more than 4,000 per cell were eliminated along with a mitochondria gene cutoff of 25%. Log normalization of aggregated reads was performed with Seurat (version 3.0.2) using a scale factor of 10,000. Feature selection was performed with the variance stabilizing transformation before integration was performed with Seurat (v3.0.1). Results: A total of 17,280 cells were recovered after filtering. Clusters were identified based on expression of unique gene markers. Differential expression analysis identified genes enriched in each population of cells corresponding to untreated versus treated libraries. Particular emphasis was given to genes enriched in monocytes. Conclusions: We report that CMP-001 induces a variety of immune responses in each cell cluster, with a unique gene signature displayed by monocytes. Human PBMCs from a healthy donor were treated with CMP-001 and anti-Qbeta for 24 hours. mRNA profiles were generated using 10X Genomics Chromium single-cell system followed by sequencing on an Illumina HiSeq4000.